The long-term objective of this project is to understand the function of human substance P (and other tachykinin receptors. Within this context, the overall focus of this project is elucidation of mechanisms involved in regulation of the substance P receptor (SPR). SPRs mediate effects of the neuropeptide substance P (SP); SP (as well as glutamate, opiates, and alpha2-adrenergic agonists) are important modulators in pain pathways. SP also plays an important role in inflammation and neurogenic edema including the axon flare reaction, vasodilation, gut motility and secretion. Given the many important physiologic roles of SP, it is important to understand SPR function. Hence, the PI plans to test the hypothesis that SPR function is regulated by receptor phosphorylation, and that SPR phosphorylation is related to agonist exposure. To achieve this goal, the PI plans to evaluate SPR phosphorylation using in vitro reconstitution (aim 1), whole cell (aim 2), and Xenopus oocyte (aim 3) systems. The PI has recently demonstrated that phosphorylation of SPRs is catalyzed by G protein receptor kinase (GRK) isozymes 2 and 3, a novel finding for receptors coupled to phosphoinositide hydrolysis. Since other GRK isozymes and protein kinase C (PKC) may also be involved in SPR phosphorylation, the first specific aim is designed to characterize phosphorylation of SPRs by various kinases. The PI has established conditions to purify and reconstitute human SPRs recombinantly expressed in Sf9 cells. This in vitro reconstitution system will be used to analyze the effect of SPR phosphorylation on interactions with G proteins and arrestins, and elucidate sites of phosphorylation using mutagenesis approaches. In order to ascertain the physiologic relevance of SPR phosphorylation, specific aim 2 uses whole cell approaches and for these studies, the PI has engineered an epitope at the amino-terminus of the SPR, and receptor specific antibodies are currently being developed. These tools will be used to assess the role of SPR phosphorylation under desensitizing conditions and conditions known to activate PKC isozymes. In specific aim 3, the role of phosphorylation in SPR function will be further analyzed using a Xenopus oocyte system. In this model, mRNA of wild type and mutant SPRs (from specific aim 1) will be transcribed in vitro, injected into oocytes, and desensitization of SP-induced chloride currents measured using a two electrode voltage clamp technique recently established in the PI's laboratory. The effect of truncated (minus carboxyl terminus) and mutated (lacking PKC phosphorylation site[s]) SPRs, and PKC modulators, on SPR desensitization will be explored. Extensive experience in receptor phosphorylation and protein biochemistry, current availability of unique substrates such as purified reconstituted SPRs, epitope tagged SPRs, and various GRK and PKC isozymes, as well as ongoing development of SPR antibodies in the PI's laboratory, places the PI in a unique position to complete the proposed studies. Information derived from these studies should facilitate understanding mechanisms underlying many SP-mediated physiologic processes.